The aim of the works presented here is to analyze the alterations induced by acute ischemia-reperfusion and chronic ischemia on mitochondrial function, in relation to alterations on heart function. Parameters of mitochondrial function were assessed on skinned fibers coming from isolated perfused rat hearts. The effects of chronic ischemia were studied on a rat model of left descending coronary artery stenosis. Two key events observed after acute ischemia-reperfusion and chronic ischemia are the decrease (or the loss) of the stimulatory effect of creatine and the alteration of outer mitochondrial permeability to cytochrome c and ADP. Taken together, these effects indicate the alteration of the intermembrane space architecture leading to the loss of intracellular adenine nucleotides compartmentation and possibly of functional coupling of mitochondrial creatine kinase and adenine nucleotide translocase. These alterations result in the impairment of intracellular energy transfer (channeling) from mitochondria to ATP-utilizing sites located in the cytosol. This may play a significant role in ischemic injury and alterations in heart function. We show that these effects were prevented by effective cardioprotective strategies like ischemic preconditioning or pharmacological preconditioning by perfusion of mitochondrial ATP-sensitive potassium channel openers. We hypothesize that an open mitochondrial ATP-sensitive potassium channel during ischemia maintains the tight structure of the intermembrane space that is required to preserve the normal low outer membrane permeability to ADP and ATP.